Project summary/abstract Sickle cell disease (SCD) patients receive transfusions for treatment and prevention of acute chest syndrome or acute stroke. Alloimmunization to red blood cell (RBC) antigens occurs more frequently in SCD leading to shortage of compatible transfusion donors. Induced pluripotent stem cells (iPSCs) can serve as a long-term source of autologous RBCs for transfusion, if they can be produced safely and in sufficient quantity. We describe a system for large-scale production of RBCs from SCD iPSCs in vitro. In this approach, iPSC-derived CD34+ hematopoietic progenitors are massively expanded via conditional expression of stem cell-specific transcription factors and differentiated into RBCs (Cell Stem Cell 13(4), 2013). iPSC-RBCs are refractory to sickling due to expression of fetal hemoglobin and do not require gene correction. We have shown that in vitro-differentiated iPSC-RBCs enucleate completely upon transfusion. We propose a scaled up bioreactor protocol to produce, irradiate, label, and track transfused human RBCs in mice. Based on our work to identify compounds that enhance RBC output in congenital anemias (Sci. Transl. Med. 9(376), 2017), we hypothesize that stimulation of autophagy can promote maturation and survival of iPSC-RBCs prior to transfusion. This work can demonstrate the feasibility of large-scale RBC production, which would benefit the most vulnerable patients with SCD.